No association of two Fas gene polymorphisms with Hashimoto's thyroiditis and Graves' disease

BACKGROUND: Apoptosis is a joint pathogenic process underlying autoimmune thyroid disease. Increased programmed cell death in thyrocytes causes hypothyroidism in Hashimoto's thyroiditis, whereas in Graves' disease infiltrating lymphocytes undergo apoptosis while thyrocytes appear to proliferate under protection of anti-apoptotic signals. The Fas/Fas ligand cascade represents a major pathway initiating apoptosis. Its role in autoimmunity is well studied and genetic polymorphisms in gene loci of Fas and its ligand have been shown to be associated with autoimmune diseases. OBJECTIVE: Due to the functional relevance of the Fas pathway in autoimmune thyroid disease we were interested in the possible contribution of polymorphisms in the Fas gene to the genetic risk of thyroid autoimmunity, which so far is mainly, but incompletely, attributed to the HLA DQ region and polymorphisms in the CTLA-4 gene. DESIGN: We genotyped Caucasian families with at least one offspring affected by Hashimoto's thyroiditis (n=95) and Graves' disease (n=109) for two Fas gene polymorphisms (g-670 G-->A in the promoter region, g-154 C-->T in exon 7). METHODS: Extended transmission disequilibrium and chi(2) testing were performed. RESULTS: Neither polymorphism alone (P=0.44 and P=0.70) nor the promoter/exon 7 haplotypes (P=0.86) were associated with Hashimoto's thyroiditis. No association with Graves' disease was observed for the promoter polymorphism (P=0.91) and exon 7 (P=0.65) or the promoter/exon 7 haplotypes (P=0.80). CONCLUSION: In summary, our data do not suggest any significant contribution of common genetic Fas variants to the genetic risk of developing Hashimoto's thyroiditis or Graves' disease

Needle Electrode-Based Electromechanical Reshaping of Cartilage

Electromechanical reshaping (EMR) of cartilage provides an alternative to the classic surgical techniques of modifying the shape of facial cartilages. The original embodiment of EMR required surface electrodes to be in direct contact with the entire cartilage region being reshaped. This study evaluates the feasibility of using needle electrode systems for EMR of facial cartilage and evaluates the relationships between electrode configuration, voltage, and application time in effecting shape change. Flat rabbit nasal septal cartilage specimens were deformed by a jig into a 90° bend, while a constant electric voltage was applied to needle electrodes that were inserted into the cartilage. The electrode configuration, voltage (0–7.5 V), and application time (1–9 min) were varied systematically to create the most effective shape change. Electric current and temperature were measured during voltage application, and the resulting specimen shape was assessed in terms of retained bend angle. In order to demonstrate the clinical feasibility of EMR, the most effective and practical settings from the septal cartilage experimentation were used to reshape intact rabbit and pig ears ex vivo. Cell viability of the cartilage after EMR was determined using confocal microscopy in conjunction with a live/dead assay. Overall, cartilage reshaping increased with increased voltage and increased application time. For all electrode configurations and application times tested, heat generation was negligible (<1 °C) up to 6 V. At 6 V, with the most effective electrode configuration, the bend angle began to significantly increase after 2 min of application time and began to plateau above 5 min. As a function of voltage at 2 min of application time, significant reshaping occurred at and above 5 V, with no significant increase in the bend angle between 6 and 7.5 V. In conclusion, electromechanical reshaping of cartilage grafts and intact ears can be effectively performed with negligible temperature elevation and spatially limited cell injury using needle electrodes

Quantitative Mass Spectrometry Evaluation of Human Retinol Binding Protein 4 and Related Variants

Background: Retinol Binding Protein 4 (RBP4) is an exciting new biomarker for the determination of insulin resistance and type 2 diabetes. It is known that circulating RBP4 resides in multiple variants which may provide enhanced clinical utility, but conventional immunoassay methods are blind to such differences. A Mass Spectrometric immunoassay (MSIA) technology that can quantitate total RBP4 as well as individual isoforms may provide an enhanced analysis for this biomarker. Methods: RBP4 was isolated and detected from 0.5 uL of human plasma using MSIA technology, for the simultaneous quantification and differentiation of endogenous human RBP4 and its variants. Results: The linear range of the assay was 7.81–500 ug/mL, and the limit of detection and limit of quantification were 3.36 ug/mL and 6.52 ug/mL, respectively. The intra-assay CVs were determined to be 5.1 % and the inter-assay CVs were 9.6%. The percent recovery of the RBP4-MSIA ranged from 95 – 105%. Method comparison of the RBP4 MSIA vs the Immun Diagnostik ELISA yielded a Passing &amp; Bablok fit of MSIA = 1.056 ELISA – 3.09, while the Cusum linearity p-value was.0.1 and the mean bias determined by the Altman Bland test was 1.2%. Conclusion: The novel RBP4 MSIA provided a fast, accurate and precise quantitative protein measurement as compared to the standard commercially available ELISA. Moreover, this method also allowed for the detection of RBP4 variants that are present in each sample, which may in the future provide a new dimension in the clinical utility of this biomarker

Dominant inhibition of Fas ligand-mediated apoptosis due to a heterozygous mutation associated with autoimmune lymphoproliferative syndrome (ALPS) Type Ib

<p>Abstract</p> <p>Background:</p> <p>Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of lymphocyte homeostasis and immunological tolerance due primarily to genetic defects in Fas (CD95/APO-1; <it>TNFRSF6</it>), a cell surface receptor that regulates apoptosis and its signaling apparatus.</p> <p>Methods:</p> <p>Fas ligand gene mutations from ALPS patients were identified through cDNA and genomic DNA sequencing. Molecular and biochemical assessment of these mutant Fas ligand proteins were carried out by expressing the mutant FasL cDNA in mammalian cells and analysis its effects on Fas-mediated programmed cell death.</p> <p>Results:</p> <p>We found an ALPS patient that harbored a heterozygous A530G mutation in the FasL gene that replaced Arg with Gly at position 156 in the protein's extracellular Fas-binding region. This produced a dominant-interfering FasL protein that bound to the wild-type FasL protein and prevented it from effectively inducing apoptosis.</p> <p>Conclusion:</p> <p>Our data explain how a naturally occurring heterozygous human FasL mutation can dominantly interfere with normal FasL apoptotic function and lead to an ALPS phenotype, designated Type Ib.</p

Concerted Regulation of cGMP and cAMP Phosphodiesterases in Early Cardiac Hypertrophy Induced by Angiotensin II

Left ventricular hypertrophy leads to heart failure and represents a high risk leading to premature death. Cyclic nucleotides (cAMP and cGMP) play a major role in heart contractility and cyclic nucleotide phosphodiesterases (PDEs) are involved in different stages of advanced cardiac diseases. We have investigated their contributions in the very initial stages of left ventricular hypertrophy development. Wistar male rats were treated over two weeks by chronic infusion of angiotensin II using osmotic mini-pumps. Left cardiac ventricles were used as total homogenates for analysis. PDE1 to PDE5 specific activities and protein and mRNA expressions were explored